Method for monitoring inhibition of platelet function

ABSTRACT

The invention provides a method of monitoring the response of platelets to a cyclooxygenase-1 (COX1) inhibitor such as aspirin. The method involves collecting platelet-containing mammalian blood treated with a COX1 inhibitor; mixing the blood with a COX1-dependent platelet agonist, such as arachidonic acid, monitoring extracellular ATP in the agonist-activated blood to generate a measurement, and comparing the measurement to a standard value. Devices, systems, and kits for carrying out the method are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/052,363, filed Mar. 20, 2008 (to issue which issued as U.S. Pat. No.7,833,793 on Nov. 16, 2010), which is a continuation of U.S. patentapplication Ser. No. 10/846,439, filed May 14, 2004 (now abandoned),both of which are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates generally to physiological analysis and moreparticularly to a method to measure inhibition of platelet function byaspirin or another cyclooxygenase-1 inhibitor.

BACKGROUND OF THE INVENTION

Millions of people are taking aspirin as therapy to reduce the risk ofheart attacks and other cardiovascular events. These include personswith elevated cholesterol, a family history of heart disease, or otherrisk factors for cardiovascular disease. Among those with risk factors,nearly all persons with implanted cardiovascular devices are at elevatedrisk of clot formation and embolization and are prescribed someanti-platelet agent, usually aspirin. In addition, many healthy peoplewithout a recognized elevated risk of cardiovascular disease also takeaspirin as a precaution.

Platelets function to stop bleeding by forming clots, and initiate theprocess of wound healing. This occurs when platelets are activated,causing them to change shape, adhere, spread, release chemicalmessengers and activators, aggregate, and assemble with fibrin.

But platelet activation and clot formation can also place a person atrisk of pathological cardiovascular events. For example, venous bloodclot formation in the legs, a condition known as deep vein thrombosis,creates the risk that the blood clots could embolize (break apart) andresult in clot entrapment in the lungs or the brain, causing pulmonaryembolisms and stroke-related conditions. Platelet activation and fibrinformation in other locations in some persons create aggregates and smallclots in the arterial circulation that can also lead to embolization andstrokes.

Each year, approximately 500,000 heart valves are implanted in theUnited States. Although biomaterial advancement has somewhat reduced therisk of thrombosis (clot formation), all patients with mechanical heartvalves are at increased risk of clot formation, embolization, andstroke, and are usually placed on aspirin therapy.

Arterial stents are another type of device placed in the circulatorysystem that place patients at risk from platelet activation. Arterialstents are placed in clogged coronary and carotid arteries to provideoxygen to cardiac tissue. They are typically around 5 mm in diameter andare made from stainless steel or other materials. Due to theintroduction of a foreign material in the blood stream, platelets canbecome activated and attach to the wall of the stented vessel. Thisleads to reocclusion (restenosis) of the stented vessel, which is a verysignificant risk in patients with arterial stents. Restenosis in thefirst 28 days is reported in 0.5 to 8% of persons receiving stents.

To reduce these and other risks of cardiovascular pathology, millions ofpatients are placed on anti-platelet drugs, most commonly aspirin.

It is useful here to briefly summarize the biochemical events ofhemostasis (the cessation of bleeding) and aspirin's role in inhibitingthe process. Normal intact vascular endothelium does not initiate orsupport platelet adhesion (although in certain vascular diseasesplatelets may adhere to intact endothelium). Vascular injury, however,exposes the endothelial surface and underlying collagen. Followingvascular injury, platelets attach to adhesive proteins such as collagenvia specific glycoproteins on the platelet surface. This adhesion isfollowed or accompanied by platelet activation, where platelets undergoa shape change from a disc shape to a spherical shape with extendedpseudopodia. At this time, the platelet release reaction also occurs.The platelets release biologically active compounds stored in thecytoplasmic bodies that stimulate platelet activation or are otherwiseinvolved in clotting reactions. These include ADP, serotonin,thromboxane A₂, and von Willebrand factor.

Following activation, glycoprotein receptors on the surface of theplatelets undergo a conformational change from a relatively inactiveconformation to an activated form. The activated receptors mediate theadhesion of more platelets by adhering to the circulating plasma proteinfibrinogen, which serves as a bridging ligand between platelets. Theadhesion and aggregation of platelets constitutes primary hemostasis.

Secondary hemostasis stabilizes the platelet mass by forming a fibrinclot. The fibrin clot is the end product of a series of reactionsinvolving plasma proteins. The process is known as blood coagulation. Incoagulation, fibrin is formed from fibrinogen, a large circulatingplasma protein, by specific proteolysis. In the process, the proteinthrombin is consumed. Fibrin monomers next spontaneously associate toform polymers and form a loose reinforcement of the platelet plug.Fibrin polymers are then cross-linked by certain enzymes. The fibrinpolymer also traps red cells and white cells to form a finished clot.

Platelets are activated by a variety of stimuli. Collagen, ADP,thrombin, and physical shear stress all activate platelets. One of thefirst steps in activation is that a platelet membrane phospholipase,phospholipase A₂, cleaves membrane lipids to release the fatty acidarachidonic acid. Arachidonic acid is oxidized in the platelet by theenzyme cyclooxygenase to the prostaglandin PGG₂. PGG₂ can beenzymatically converted to PGH₂, and PGH₂ is converted by thromboxanesynthetase to thromboxane A₂ (TxA₂).

TxA₂ is a very potent activator of platelets and greatly amplifies theplatelet release reaction, where the platelets secrete the contents ofcertain cytoplasmic bodies, including alpha granules and dense bodies.Among the components secreted from dense bodies are ADP, Ca⁺⁺, Mg⁺⁺, andserotonin.

Aspirin acts by acetylating and inactivating cyclooxygenase-1 inplatelets, preventing the synthesis of TxA₂. By preventing the synthesisof TxA₂, aspirin significantly reduces platelet activation and thusreduces clotting. Aspirin inactivates cyclooxygenase-1 (COX1) at a lowerdose and more completely than it inactivates or inhibits another isoformof cyclooxygenase, cyclooxygenase-2. COX1 is the predominantcyclooxygenase in platelets. COX2 is involved in inflammation. (Vane, J.R., et al., 2003, The mechanism of action of aspirin, ThrombosisResearch 110: 255.)

Thus, by inhibiting platelet activation, aspirin for most patients is aneffective agent to prevent clots and pathological cardiovascular events.But many people are resistant to aspirin. In one study, 5.5% or 9.5% ofpatients were resistant to aspirin, as assayed by two differenttechniques, and 23.8% of patients were semi-resistant (Gum, P. A., etal., 2001, Am. J. Cardiology 88: 230). Other studies estimate 5-40% ofpatients are aspirin resistant, depending on the assay and thepopulation studied (Bhatt, D. L., 2004, J. Am. College of CardiologyVol. 43, No. 6, 2004). This is very important, because aspirinresistance is significantly associated with an increased risk of death,myocardial infarction, or cerebrovascular accident (Altman, R., et al.,2004, Thrombosis J. 2: 1).

It is important to identify patients resistant to aspirin or other COX1inhibitors, because if they are identified they can be placed on otherplatelet inhibitors that act by a different mechanism. This is importantnot only for proper treatment of the patients, but also for costsavings. The other platelet inhibitors are much more expensive thanaspirin, so it would be extremely expensive to indiscriminatelyprescribe them. (Other platelet inhibitors include ADP inhibitors suchas ticlopidine, and monoclonal antibodies that block the GPIIbIIIareceptor such as RHEOPRO.) Physicians are only likely to prescribe themwhen it can be shown that aspirin is not working.

Various techniques have been used to measure platelet function andaspirin resistance. Among these is platelet aggregation. In thistechnique, platelet-rich plasma was prepared from whole blood. ADP andarachidonic acid were added to activate the platelets. And aggregationof the platelets was measured by optical density changes. (Gum, P. A.,et al., 2001, Am. J. Cardiology 88: 230.) Another technique uses adevice named the platelet function analyzer-100 (PFA-100). The PFA-100uses a disposable cartridge with an aperture cut into a collagen-coatedmembrane infused with either ADP or epinephrine. Whole blood(approximately 1 ml) is pumped through the aperture at high shear rate.The blood comes into contact with the membrane where platelets adhereand aggregate. A platelet plug forms, occluding the aperture andstopping blood flow. The closure time is a measure of platelet function.(Gum, P. A., et al., 2001, Am. J. Cardiology 88: 230.) Another deviceused to measure aspirin response is the Accumetrix VERIFYNOW AspirinAssay (www.accumetrics.com/products/ultegra_asa.html). This product usesa turbidity-based optical detection system. The device containsfibrinogen-coated beads, and a platelet agonist. Blood is withdrawn,citrated, and then mixed with the coated beads and the agonist.Aggregation of the platelets to the beads is measured optically.

Prior tests for aspirin response have various drawbacks. Many usesignificant volumes of blood. Some require time-consuming andlabor-consuming processing of the blood. And some measure adhesion andaggregation of the platelets, which are complex phenomena that are theend result of several interacting steps, rather than more directlymeasuring steps more directly related to aspirin's inhibition ofcyclooxygenase.

A new method of monitoring aspirin response or response to other COX1inhibitors is needed. Preferably, the method would use a small volume ofblood (e.g., less than a drop), use unprocessed whole blood, be fast,and be relatively specific for the pathway inhibited by aspirin, theCOX1 pathway.

BRIEF SUMMARY OF THE INVENTION

The invention involves a method to measure inhibition of plateletfunction by aspirin or another cyclooxygenase-1 inhibitor. Blood iswithdrawn from a patient who has taken aspirin and mixed with aCOX1-dependent platelet agonist, typically arachidonic acid, which isthe substrate for the enzyme cyclooxygenase-1 in platelets. Arachidonicacid is converted in platelets by COX1 to PGG₂ and then to thromboxaneA₂. Thromboxane A₂ is a potent activator of platelets and instigates theplatelet release reaction, where the platelets secrete the contents oftheir dense bodies and alpha granules. One of the compounds secretedfrom the dense bodies is ATP. In the present method, the secretedextracellular ATP or other extracellular platelet-release-reaction onproduct is detected, and its concentration is quantitatively orqualitatively determined. The result from the determination of theextracellular release-reaction product (e.g., ATP) concentration iscompared with a standard, which can be the result from blood taken fromthe same patient before she or he took aspirin.

Aspirin acts by inactivating cyclooxygenase-1, the predominantcyclooxygenase in platelets. Thus, if aspirin has inactivated most orall cyclooxygenase-1, the amount of thromboxane A₂ produced will bedecreased, the release reaction will be weaker, and less releasereaction product (e.g., ATP) will be secreted, as compared to when thepatient does not take aspirin.

The method can use less than a drop of blood. Whole blood can be usedwithout any further processing. And results can be obtained at thepatient's bedside in less than five minutes. In addition, since ATPrelease is strongly induced by thromboxane A₂, the assay has highspecificity for the pathway inhibited by aspirin, the COX1 pathway:thromboxane A₂ synthesis from arachidonic acid.

In one embodiment, a finger prick is performed to draw a drop of bloodfrom a patient who has not had aspirin or another COX 1 inhibitor for atleast several hours, preferably 8 days. (The lifetime of platelets isabout 8 days. Aspirin covalently modifies the COX1 in platelets toinactivate it. So the effect of aspirin lingers until all the plateletsinactivated by aspirin have been cleared. Other COX1 inhibitors may actby a noncovalent inhibition and have a shorter duration of action.)Approximately 10 μl of blood is taken and diluted 500:1 with buffer. TheATP-consuming and light-producing enzyme luciferase and its substrateluciferin are added, and a background reading of light emission istaken. Arachidonic acid is then added to the sample, and the increase inlight emission is monitored. The patient then swallows aspirin, and theprocedure is performed again, and the results compared. If the aspirinis working, there will be a large decrease in light emission in theblood sample drawn after the patient takes aspirin as compared to thesample drawn before the patient takes aspirin. If the patient isaspirin-resistant, the difference between the two results will besmaller.

Thus, one embodiment of the invention provides a method of monitoringCOX1-inhibitor response involving: (a) collecting platelet-containingmammalian blood treated with a COX1 inhibitor; (b) mixing the blood witha COX1-dependent platelet agonist to generate agonist-activated blood;(c) monitoring an extracellular platelet-release-reaction product (e.g.ATP) in the agonist-activated blood to generate a measurement; and (d)comparing the measurement to a standard value.

Another embodiment of the invention provides a device for monitoringCOX1-inhibitor response including: (a) a fluid-tight material forming anassay chamber; (b) a pump functionally linked to the assay chamber forpumping fluids into the assay chamber; (c) a light detector functionallylinked to the assay chamber for detecting light emitted in the assaychamber; (d) a fluid-tight material forming a blood chamber for holdingblood, the blood chamber functionally linked to the pump; (e) afluid-tight material forming an enzyme chamber for holding a solution ofa light-producing ATP-consuming enzyme, the enzyme chamber functionallylinked to the pump; and (f) a fluid-tight material forming an agonistchamber for holding a solution of a COX1-dependent platelet agonist, theagonist chamber functionally linked to the pump.

Another embodiment of the invention provides a system for monitoringCOX1-inhibitor response including: (a) a fluid-tight material forming anassay chamber; (b) a pump functionally linked to the assay chamber forpumping fluids into the assay chamber; (c) a light detector functionallylinked to the assay chamber for detecting light emitted in the assaychamber; (d) a fluid-tight material forming a blood chamber for holdingblood, the blood chamber functionally linked to the pump; (e) afluid-tight material forming an enzyme chamber for holding a solution ofa light-producing ATP-consuming enzyme, the enzyme chamber functionallylinked to the pump; (f) a fluid-tight material forming an agonistchamber for holding a solution of a COX1-dependent platelet agonist, theagonist chamber functionally linked to the pump; (g) a controllercoupled to the pump and programmed to deliver to the assay chamber apredetermined volume of the blood, a predetermined volume of thelight-producing enzyme solution, and a predetermined volume of theagonist solution; and (h) a display operably coupled to the photoncounter for displaying results from the light detector.

Another embodiment of the invention provides a kit for determiningresponse to a COX1 inhibitor containing: a COX1-dependent plateletagonist; an ATP-consuming enzyme; and instruction means indicating thatthe enzyme and the agonist are to be used to determine a subjectmammal's physiological response to a COX1 inhibitor.

Another embodiment of the invention provides a kit for determiningresponse to a COX1 inhibitor containing: (1) a device for monitoringCOX1-inhibitor response comprising: (a) a fluid-tight material formingan assay chamber; (b) a pump functionally linked to the assay chamberfor pumping fluids into the assay chamber; (c) a light detectorfunctionally linked to the assay chamber for detecting light emitted inthe assay chamber; (d) a fluid-tight material forming a blood chamberfor holding blood, the blood chamber functionally linked to the pump;(e) a fluid-tight material forming an enzyme chamber for holding asolution of a light-producing ATP-consuming enzyme, the enzyme chamberfunctionally linked to the pump; and (f) a fluid-tight material formingan agonist chamber for holding a solution of a COX1-dependent plateletagonist, the agonist chamber functionally linked to the pump; and (2)instruction means indicating that the device is to be used to determinea subject mammal's physiological response to a COX1 inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the pathway of thromboxane A₂ synthesis from arachidonicacid.

FIGS. 2A-C show luciferase assay results with arachidonic-acid-treateddiluted blood drawn from three individuals before and after takingaspirin. The light emission reflects extracellular ATP.

FIG. 3 is a schematic diagram of a device for monitoring COX1-inhibitorresponse.

FIG. 4 is a schematic diagram of a device for monitoring COX1-inhibitorresponse, where the device includes a valve.

FIG. 5 is a schematic diagram of a system for monitoring COX1-inhibitorresponse.

FIG. 6 is a diagram of an apparatus for monitoring aspirin response thatpumps fluids into a cuvette.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “COX1 inhibitor” refers to an agent that selectively inhibitsCOX1 more than it inhibits COX2 or inhibits COX1 at a lowerconcentration of agent than COX2. The inhibition can be due to covalentor noncovalent interactions. The inhibition can be inactivation,competitive, uncompetitive, or non-competitive inhibition. The mostcommon COX1 inhibitor is aspirin. Another example of a COX1 inhibitor isSC-58560 (Bolla, M., et al., Hypertension, Apr. 19, 2004).

The term “platelet-containing blood” refers to whole blood or processedblood that contains platelets, e.g., platelet-rich plasma.

The terms “measurement” and “standard value” refer to quantitative orqualitative assessments that can be compared with each other.

The term “blood treated with a COX1 inhibitor” refers to blood contactedwith a COX1 inhibitor either in vivo or ex situ. The blood can becontacted with the inhibitor (e.g., aspirin), e.g., by orallyadministering aspirin to the mammal, or by contacting blood with aspirinafter withdrawing the blood from the mammal.

The term “dilution medium” refers to any suitable liquid for dilutingblood. This typically is an aqueous solution that includes a pH bufferand solutes sufficient to maintain an appropriate osmoticum in thedilution buffer, such as phosphate buffered saline or HEPES/HANKS.Dilution medium, however, need not contain a pH buffer, and could bewater or for instance, a salt or sucrose solution in water.

The term “ATP-consuming enzyme” refers to an enzyme that uses andconsumes ATP as a substrate.

The term “chamber” refers to a vessel that contains fluids. The chambercan be substantially enclosed, or could be, for instance, a section oftubing in which liquid is held.

A “light detector” as used herein is any apparatus or component of anapparatus capable of qualitatively or, preferably, quantitativelydetermining the intensity of light or number of photons emitted in anassay chamber. An example of a light detector is a photon counter.

A “COX1-dependent platelet agonist” as used herein is an agonist whoseactivation of platelets is substantially inhibited by aspirin inaspirin-sensitive humans. Preferably, a COX1-dependent platelet agonistis an agonist that induces a maximal rate of extracellular ATP releasein aspirin-sensitive humans in the assays described herein that is atleast 5 times higher in non-aspirin-treated blood than inaspirin-treated blood. Conversely, a platelet agonist that is not COX1dependent as used herein refers to an agonist whose activation ofplatelets is not substantially inhibited by aspirin in aspirin-sensitivehumans. It induces a maximal rate of extracellular ATP release inaspirin-sensitive humans in the assays described herein that is lessthan 5 times higher, preferably less than 3 times higher, morepreferably less than 2 times higher, most preferably approximately thesame, in non-aspirin-treated blood than in aspirin-treated blood.

The most preferred COX1-dependent platelet agonist is arachidonic acid.The most preferred platelet agonist that is not COX1 dependent isthromboxane A₂. Other platelet agonists that are not COX1 dependentinclude collagen, thrombin, and ADP.

“An extracellular platelet-release-reaction product,” as used herein, isa component secreted from platelets in response to arachidonic acid,whose maximal rate of secretion in aspirin sensitive humans is at least2 times faster before the person takes aspirin than the maximal rate ofsecretion after the person takes aspirin. Preferably, theplatelet-release-reaction product is a product whose maximal rate ofsecretion is at least 3 times faster, more preferably at least 5 timesfaster, before the person takes aspirin than after the person takesaspirin. The extracellular platelet-release-reaction product may besoluble in the extracellular medium or may become exposed on theextracellular platelet membrane surface after the release reaction.

The preferred extracellular platelet-release-reaction product is ATP.Other examples of extracellular release reaction products include thedense body components GDP, GTP, ADP, serotonin, Mg⁺⁺, and Ca⁺⁺, as wellas the alpha granule components fibrinogen, von Willebrand factor,fibronectin, thrombospondin, vitronectin, factor V, factor XI, proteinS, platelet-derived growth factor, transforming growth factor β,epidermal growth factor, P-selectin, GMP 33, and osteonectin. (TheMegakaryocyte-Platelet System, R. R. Rifkin, in Clinical Hematology:Principles, Procedures, Correlations, second edition, Stiene-Martin, E.A., et al., eds., Lippincott, N.Y., 1998.)

Description

The pathway of thromboxane A₂ synthesis from arachidonic acid inplatelets is shown in FIG. 1. Aspirin inhibits this pathway byacetylating cyclooxygenase and inactivating the enzyme.

In one embodiment of the method of monitoring COX1-inhibitor response,the COX1 inhibitor is aspirin.

In one embodiment, the extracellular platelet-release-reaction productis ATP.

In one embodiment, the COX1-dependent platelet agonist is arachidonicacid.

Another example of a class of COX1-dependent platelet agonists isphospholipase A₂ activators. Compounds that activate phospholipase A₂stimulate the release of arachidonic acid by the phospholipase A₂ inplatelets.

Other specific examples of COX1-dependent platelet agonists includepropyl gallate and U-46619 (Bolla, M. et al. Hypertension, Apr. 19,2004.)

In one embodiment of the method of monitoring COX1-inhibitor response,the step of monitoring extracellular ATP involves adding to the blood anATP-consuming enzyme that catalyzes a reaction, and monitoring theenzyme-catalyzed reaction.

In one embodiment, monitoring the enzyme-catalyzed reaction involvesmonitoring a product produced by the reaction. The product can be, forinstance, light. In other embodiments, monitoring the reaction involvesmonitoring the disappearance of a reactant.

In one embodiment of the method using an enzyme, the enzyme isluciferase (e.g., firefly luciferase). In particular embodiments, themethod further involves adding luciferin to the blood. In oneembodiment, the luciferin is firefly luciferin.

The steps of the invention can be performed in any suitable order. Forinstance, in the embodiments that involve adding to the blood anATP-consuming enzyme that catalyzes a reaction and monitoring thereaction, the enzyme can be added to the blood before, together with, orafter the COX1-dependent agonist. Likewise, where another enzymesubstrate such as luciferin is added, the substrate can be added before,together with, or after both the enzyme and the agonist.

In one particular embodiment, luciferin and luciferase are added to theblood before the blood is mixed with the COX1-dependent plateletagonist. In another particular embodiment, luciferin and luciferase areadded to the blood after the blood is mixed with the agonist.

Particular embodiments of the method of the invention further involvediluting the blood.

In a particular embodiment where the blood is diluted, the step ofmonitoring extracellular ATP involves adding luciferin and luciferase tothe blood, and monitoring light produced by the luciferase.

One of the advantages of the method of the invention is that smallvolumes of blood can be used. Blood can be collected by a finger prick,and less than a drop can be used. In particular embodiments, less than120, less than 100, less than 75, less than 40, less than 20, less than12, or less than 10 microliters are collected from the mammal. In otherparticular embodiments, less than 120, less than 100, less than 75, lessthan 40, less than 20, less than 12, or less than 10 microliters ofblood is mixed with the COX1-dependent platelet agonist.

In particular embodiments, the mammal is a human.

In particular embodiments of the invention, the platelet-containingblood is whole blood.

One method of determining the standard value for comparison in the assayis to collect non-aspirin-treated blood (or blood not treated with aCOX1 inhibitor) from the same mammalian subject, and perform the sameassay on the non-aspirin-treated blood to generate a measurement to beused as the standard value. In these embodiments, the standard value isdetermined by a method involving collecting platelet-containing standardblood from the mammal; mixing the standard blood with the COX1-dependentplatelet agonist to generate agonist-activated standard blood; andmonitoring the extracellular platelet-release-reaction product (e.g.,ATP) in the agonist-activated standard blood to generate the standardvalue; wherein the standard blood is not treated with the COX1inhibitor. Preferably, the mammalian subject has not been treated withthe COX1 inhibitor for at least 3 hours, more preferably at least 6hours, more preferably at least 12 hours, and more preferably still atleast 24 hours, and more preferably still at least 8 days before theblood is collected. If the COX1 inhibitor is aspirin, preferably thesubject has not been treated with aspirin for at least 24 hours, morepreferably for at least 3 days, more preferably still for at least 8days before the blood is collected.

The standard value can be obtained also by assaying blood of subjectsdifferent from the subject whose COX1-inhibitor-treated blood isassayed, and establishing a typical measured value reflecting theextracellular platelet-release-reaction product in agonist-activatedblood where the blood is not COX1-inhibitor treated or where the bloodis from subjects either resistant or sensitive to the COX1 inhibitor.

The measurement and standard value can be quantitative or qualitative,and can be time courses or individual time points. For instance, themeasurement and standard value can be direct measurements ofextracellular ATP concentration at a certain time point after mixing theblood with the agonist. They can also be time courses of light emissionusing luciferin or luciferase or individual time points of quantity oflight emitted at a specific time after agonist mixing using luciferinand luciferase.

Another potential measurement is to determine the amount ofextracellular platelet-release-reaction product (e.g., ATP) secreted dueto the agonist, then lyse the platelets with detergent such as TRITONX-100 or SDS, and determine the extracellular platelet-release-reactionproduct after platelet lysis, which represents the total cellularproduct. The measurement and standard value can be the difference inproduct (e.g., ATP) concentration before and after detergent lysis, orthe ratio of product concentration before cell lysis to productconcentration after cell lysis. That difference or ratio measurement canbe compared between blood treated with the COX1 inhibitor (themeasurement) and blood not treated with the COX1 inhibitor (thestandard).

In a particular embodiment, the standard value is determined by a methodinvolving: (a) collecting platelet-containing standard blood from themammal; (b) diluting the standard blood; (c) mixing the standard bloodwith the COX1-dependent platelet agonist to generate agonist-activatedstandard blood; and (d) monitoring extracellular ATP in theagonist-activated standard blood to generate the standard value, whereinthe step of monitoring extracellular ATP involves (i) adding luciferinand luciferase to the standard blood, and (ii) monitoring light producedby the luciferase; wherein the standard blood is not treated with theCOX1 inhibitor.

In a particular embodiment of that method, luciferin and luciferase areadded to the standard blood before mixing the standard blood with theagonist (e.g., arachidonic acid). In another embodiment, they are addedafter mixing the standard blood with agonist.

Thus, in one embodiment, a result of assaying for the extracellularplatelet-release-reaction product (e.g., ATP) in agonist-activatedCOX1-inhibitor-treated blood is compared with a result obtained usingblood not treated with the COX1 inhibitor, preferably from the sameindividual.

In one embodiment, a result of assaying for the extracellularplatelet-release-reaction product in arachidonic-acid-activatedaspirin-treated blood is compared with a result obtained usingnon-aspirin-treated blood, preferably from the same individual.

The results from COX1-dependent agonist treatment can also be comparedto results obtained using an agonist that is not COX1 dependent. Forinstance, the amount of extracellular platelet-release-reaction productin arachidonic-acid-activated COX1-inhibitor-treated blood can becompared with the amount of extracellular platelet-release-reactionproduct in thromboxane-A₂-activated COX1-inhibitor-treated blood.Thromboxane A₂ is the end product of the cyclooxygenase-1 pathway, thetarget of aspirin. So thromboxane A₂ will activate platelets to the sameextent whether or not they are treated with a COX1 inhibitor such asaspirin and whether or not the platelets are sensitive to the COX1inhibitor. In contrast, arachidonic acid will activate platelets to amuch greater extent if the platelets are not treated with the COX1inhibitor or are insensitive to the COX1 inhibitor.

Likewise, aspirin is also not expected to have as large an effect on theextent of platelet activation by certain other activators, includingthrombin, collagen, and ADP, as it does on the extent of plateletactivation by arachidonic acid. Thus, the amount of extracellular ATPinduced by arachidonic acid activation can be compared to the amount ofextracellular ATP induced by activation with thrombin, collagen, or ADP.

Accordingly, in certain embodiments of the invention, the standard valueis determined by a method involving: collecting platelet-containingCOX1-inhibitor-treated standard blood from the mammal; mixing thestandard blood with a non-COX1-dependent platelet agonist to generateactivated standard blood; and monitoring the extracellularplatelet-release-reaction product (e.g., ATP) in the activated standardblood to generate the standard value. The non-COX1-dependent plateletagonist can be, for instance, thromboxane A₂, thrombin, collagen, orADP.

Where the extracellular platelet-release-reaction product is ATP, apreferred method of detecting the ATP is by enzymatic methods, asdescribed herein, in particular by the use of luciferin. ATP can also bedetected, for example, by HPLC or gas chromatography, with or withoutmass spectrometry. Other extracellular platelet-release-reactionproducts can also be detected by HPLC, gas chromatography, and/or massspectrometry. Some platelet-release-reaction products can be detected byenzymatic means, where for instance the release reaction product is asubstrate for an enzymatic reaction, and the course of the enzymaticreaction is monitored by monitoring a product of the enzymatic reaction.Some extracellular platelet-release-reaction products, particularlyproteins, are amenable to detection by immunologic means, such as ELISA.

The invention also provides a device, schematically shown in FIG. 3,having a fluid-tight material forming an assay chamber 11, a pump 20 forpumping fluids into the assay chamber, and a light detector 12 fordetecting light emitted in the assay chamber. The device also includes afluid-tight material forming a blood chamber 14 for holding blood 15,the blood chamber functionally linked to the pump 20. The device alsoincludes a fluid-tight material forming an enzyme chamber 16 for holdinga solution of a light-producing enzyme 17, the enzyme chamberfunctionally linked to the pump 20. The device also includes afluid-tight material forming an agonist chamber 18 for holding asolution of a COX1-dependent platelet agonist 19, the agonist chamberfunctionally linked to the pump 20. The “solution” of a COX1-dependentplatelet agonist can be pure agonist or undissolved agonist insuspension, but preferably is a true solution of the agonist (e.g.,arachidonic acid) dissolved in water, ethanol, or another solvent.

In one embodiment, the device further includes a valve 13 functionallylinked to the pump, the assay chamber, the blood chamber, the enzymechamber, and the agonist chamber (FIG. 4). The valve can be, forinstance, a multi-position valve.

In one embodiment, the device further includes a fluid-tight materialforming a dilution medium chamber for holding dilution medium, thedilution medium chamber functionally linked to the pump.

Two or three of the assay chamber, the blood chamber, the enzymechamber, and the agonist chamber can be the same chamber.

In one embodiment, all of the four chambers—the assay chamber, the bloodchamber, the enzyme chamber, and the agonist chamber—are separatechambers.

The invention also provides a system for monitoring aspirin responseinvolving all the elements of the device, along with a controller 21 fordelivering to the assay chamber a predetermined volume of the blood, apredetermined volume of the light-producing enzyme solution, and apredetermined volume of the agonist solution; and a display 23 linked tothe light detector for displaying light detector results (FIG. 5).

In particular embodiments, the predetermined volume of blood is lessthan 120, less than 100, less than 75, less than 40, less than 20, lessthan 12, or less than 10 microliters.

One embodiment of the invention provides a kit for determining responseto a COX1 inhibitor (e.g., aspirin) involving a COX1-dependent plateletagonist (e.g., arachidonic acid), an ATP-consuming enzyme, andinstruction means.

In one embodiment of the kit, the enzyme is a light-producingATP-consuming enzyme.

The invention will now be illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Assaying Platelet Aspirin Response

Methods:

Volunteers who had not taken aspirin in the previous 24 hours underwenta finger prick to draw blood. Blood (2 μl) was diluted into 1 ml ofbuffer (PBS, 0.9% NaCl, 10 mM sodium phosphate, pH 7.0) in a clear testtube. An aliquot (15 μl) of luciferin/luciferase solution (containing 10mg/ml luciferin and 10 μg/ml luciferase) as added to the diluted blood.The solution was mixed and placed in a Zylux FEMPTOMASTER FB12 photoncounter. A baseline reading of the light emission was taken. The testtube was then withdrawn, and 5 μl of 15 mM arachidonic acid was added tothe solution and the solution was mixed. The test tube was thenreinserted in the photon counter and light emission readings were takenfor approximately 2 to 4 minutes.

The volunteer then swallowed or chewed an aspirin (325 mg). A bloodsample was taken, and the process was repeated. The results from blooddrawn from an individual before and after taking aspirin were compared.

Results:

Time courses of light emission in the assays for three individualsbefore and after taking aspirin are shown in FIGS. 2A-C. In FIGS. 2A and2C the slope of light emission vs. time after arachidonic acid is addedto the sample is very steep for the no-aspirin sample but almost flatfor the aspirin sample. The ratio of the no-aspirin maximal slope to theaspirin maximal slope is greater than 10:1. In contrast, results for theindividual of FIG. 2B show a ratio of maximal slopes of the no-aspirinto the aspirin sample of slightly less than 2:1. Thus, the individual ofFIG. 2B is less responsive to aspirin than the individuals of FIGS. 2Aand 2C.

Discussion:

A very small amount of blood was used in this assay—2 μl—easily lessthan a drop of blood. Even less could be used. The limiting factor forusing smaller volumes would probably be inaccuracy of measuring thevolumes, which would cause imprecision in the assay results.

Lesser amounts of luciferin, luciferase, and arachidonic acid could beused than were used in this example. With lower amounts of luciferin orluciferase, the light emission would plateau sooner and at lower levels.The inventor believes that instead of the 15 mM arachidonic acidsolution used in this example, 10 μM or less and possibly 1 μM or lessarachidonic acid could be used without affecting the results.

To determine aspirin response, the data can be analyzed in several ways.The maximal slopes of the no-aspirin and aspirin samples can becompared. The peak light emission levels can be compared. The lightemission at a certain time after adding arachidonic acid to the blood,e.g., 2 minutes, can be compared. Or a difference in light emissionlevels can be calculated for each sample and then compared, e.g., lightemission at 2 minutes after adding arachidonic acid minus emission at 10seconds after adding arachidonic acid or minus emission before addingarachidonic acid.

Example 2 An Apparatus for Monitoring Aspirin Response that Pumps Fluidsinto a Cuvette

The apparatus is shown in FIG. 6. Blood is diluted and stored in adiluted blood chamber or container 14. Firefly luciferin and luciferaseare stored in a firefly extract container 16. Arachidonic acid is storedin a third container, an arachidonic acid container 18. Tubing 23connects the solutions in each container to a bi-directional pump 20.The tubing to the three containers merges together before the pump, sothat fluid is pumped by the pump simultaneously from all threecontainers and mixed together. If the tubing to each container is thesame diameter, equal volumes are pumped from each container. By usingtubing of unequal diameters to each container, the volume drawn fromeach container relative to the others can be controlled. The drawnfluids come together in the merged section of the tubing and are pulledby the pump 20 past a check valve 13. After the check valve the fluidsare pumped by the bidirectional pump to a reaction cuvette 11 positionedwithin a photon counter 12 such as a photomultiplier tube. In thereaction cuvette 11, the fluids are further mixed, either by brownianmotion or by a mechanical mixer, and the photons emitted are counted bythe photon counter.

The pump and photon counter are preferably microprocessor controlled.

Example 3 Fluid Injection Analysis and Sequential Injection Analysis

A typical fluid injection analysis (FIA) manifold includes a pump,injection valve, photon counter, and tubing. The pump is used to propelone or more streams through the photon counter via a narrow-bore(0.5-0.8 mm ID) tubing. These streams may be reagents or buffer. Theinjection valve is used to periodically introduce a small volume(generally less than 100 μl) sample of one reagent into the carrierstream. As the sample is carried to the detector, the fluid dynamics offlow through a narrow-bore tubing mixes sample and carrier, leading tothe reaction to form a detectable species (in this case light emission).This species is sensed by the detector as a transient peak. The heightand area of the peak are proportional to concentration and are used toquantify the concentration of the species being determined by comparisonto samples of known concentration (a calibration curve). Thus, forinstance, blood, dilution buffer, and luciferin/luciferase can bepremixed in the carrier stream, and arachidonic acid can be theinjectable reagent added last to start the reaction.

In sequential injection analysis, a selection valve and a bidirectionalpump are used to draw up small volumes of sample and reagents, and thenpropel them through a coil to a detector. Again the process causesmixing of the sample and reagent segments leading to chemistry thatforms a detectable species before reaching the detector.

A type of sequential injection analysis device that could be adapted foruse in the invention is described in U.S. Pat. No. 6,716,391.

Information and supplies for fluid injection analysis and sequentialinjection analysis are available at www.globalfia.com, Global FIA, Inc.,Fox Island, Wash.

All cited patents, patent-related documents, and other references arehereby incorporated by reference.

What is claimed is:
 1. A method of monitoring inhibition of plateletfunction response comprising: collecting less than 100 μl ofplatelet-containing mammalian test blood from a patient treated with aplatelet inhibitor; mixing the test blood with a platelet agonist togenerate agonist-activated test blood; monitoring an extracellularplatelet-release-reaction light emission in the agonist-activated testblood to generate a test measurement of the light emission; comparingthe measured test light emission to a standard value, wherein thestandard value of the light emission is determined by collectingplatelet-containing standard blood from the patient; mixing the standardblood with the platelet agonist to generate agonist-activated standardblood; and monitoring an extracellular platelet-release-reaction lightemission in the agonist-activated standard blood to generate thestandard value, wherein the standard blood is collected from the patientbefore the patient is treated with the platelet inhibitor; anddetermining an inhibitory effect of platelet activation based on thecomparison of the measured test light emission to the standard value. 2.The method of claim 1, wherein the platelet inhibitor is aspirin.
 3. Themethod of claim 1, wherein the extracellular platelet-release-reactionlight emission is based on the amount of ATP.
 4. The method of claim 3,wherein the monitoring of the light emission comprises: adding to theblood an ATP-consuming enzyme that catalyzes a reaction; and monitoringlight emission from the enzyme-catalyzed reaction.
 5. The method ofclaim 4, wherein the enzyme is luciferase, the method further comprisingadding luciferin to the blood.
 6. The method of claim 5, wherein theluciferin and luciferase are added to the blood before the blood ismixed with the platelet agonist.
 7. The method of claim 5, wherein theluciferin and luciferase are added to the blood after the blood is mixedwith the platelet agonist.
 8. The method of claim 3, wherein less than10 μl of blood is collected.
 9. The method of claim 3, wherein theplatelet-containing blood is whole blood.
 10. The method of claim 3,further comprising: diluting the blood, wherein the monitoring of thelight emission comprises: adding luciferin and luciferase to the blood;and monitoring light produced by the luciferase.
 11. The method of claim10, wherein the standard value of the light emission is determined by amethod comprising: collecting less than 100 μl of platelet-containingstandard blood from the mammal; diluting the standard blood; mixing thestandard blood with the platelet agonist to generate agonist-activatedstandard blood; and monitoring extracellular ATP in theagonist-activated standard blood to generate the standard value, whereinthe monitoring of the ATP comprises (a) adding luciferin and luciferaseto the standard blood, and (b) monitoring light emission produced by theluciferase; wherein the standard blood is not treated with the plateletinhibitor.
 12. The method of claim 11, wherein the platelet agonist isarachidonic acid.
 13. The method of claim 1, wherein the plateletagonist is arachidonic acid.
 14. The method of claim 1, wherein theplatelet agonist is a phospholipase A₂ activator.
 15. The method ofclaim 1, further comprising: diluting the blood.
 16. The method of claim15, wherein the agonist-activated blood is diluted enough to preventplatelet aggregation.
 17. The method of claim 15, wherein theagonist-activated blood is diluted at least 1/20.
 18. The method ofclaim 1, wherein the standard value of the light emission is determinedby a method comprising: collecting less than 100 μl ofplatelet-containing standard blood from the mammal; mixing the standardblood with the platelet agonist to generate agonist-activated standardblood; and monitoring the extracellular platelet-release-reaction lightemission in the agonist-activated standard blood to generate thestandard value, wherein the standard blood is not treated with theplatelet inhibitor.
 19. A method for monitoring inhibition of plateletfunction response comprising: providing a predetermined volume of asolution of light-producing ATP-consuming enzyme; providing apredetermined volume of a solution of platelet agonist; providing anassay chamber; collecting a test volume of less than 100 μl ofplatelet-containing mammalian blood from a patient treated with aplatelet inhibitor; delivering and mixing the predetermined volume ofthe solution of light-producing ATP-consuming enzyme, the predeterminedvolume of the solution of platelet agonist, and the test volume of bloodinto the assay chamber; detecting an amount of test light in the assaychamber that is emitted from the mixed solutions and the test volume ofblood; comparing the detected amount of test light emission to astandard value of light emission, wherein the standard value of lightemission is determined by collecting platelet-containing standard bloodfrom the patient, mixing the standard blood with the platelet agonist togenerate agonist-activated standard blood, and monitoring anextracellular platelet-release-reaction light emission in theagonist-activated standard blood to generate the standard value, whereinthe standard blood is collected from the patient before the patient istreated with the platelet inhibitor; and determining an inhibitoryeffect of platelet activation based on the comparison of the measuredtest light emission to the standard value.